Protein kinases constitute the largest family of human enzymes. As principal regulators of signal transduction pathways, protein kinases play a critical role in a wide range of cellular processes such as proliferation, differentiation and apoptosis. Protein kinases are currently considered to be the largest class of proteins amenable to therapeutic intervention by small molecule drugs (Cohen, P., 2002, Nat. Rev. Drug Discov. 1:309-315; Hopkins, A. L. and Groom, C. R., 2002, Nat. Rev. Drug Discov. 1:727-730). With the advent of genome databases of clinically important protozoan parasites, it is clear that the evolutionary distance between essential, protozoan protein kinases and the host organisms' corresponding kinase orthologues presents many opportunities for chemotherapeutic intervention of protozoan parasitic proliferation (Doerig, C. et al., 2002, Trends Parasitol. 18:366-371). Such differences have been successfully exploited in the case of the coccidian parasite cGMP-dependent protein kinase (“PKG”) for which a selective, small molecule inhibitor has been identified that effectively controls parasite proliferation, preventing coccidiosis caused by Eimeria species in poultry and Toxoplasmosis caused by Toxoplasma gondii in a mouse model (Gurnett, A. M. et al., 2002, J. Biol. Chem. 277:15913-15922; Nare, B. et al., 2002, Antimicrob. Agents Chemother. 46:300-307; Donald, R. G. K. et al., 2002, Eukaryot. Cell. 1:317-328). Knockaert et al. (2000, Chem. Biol. 7:411-422) also demonstrated the exploitable differences between parasite kinases and their host cell counterparts using affinity chromatography with an immobilized inhibitor of cyclin-dependent protein kinase (“CDK”), purvalanol B. While the ligand used in these studies is an exceptionally potent and selective inhibitor of mammalian CDKs, affinity chromatography of parasite lysates, including Plasmodium falciparum, Leishmania mexicana, Toxoplasma gondii and Trypanosoma cruzi, with this inhibitor showed protozoan casein kinase I (“CKI”) proteins, rather than CDK enzymes, tightly bound to the column. In contrast, parallel affinity chromatography of extracts of marine invertebrates and some vertebrate tissues positively identified associated CDKs without selection of CKI enzymes.
Casein kinase I enzymes represent a family of multipotential serine/threonine proteins kinases common to all eukaryotic cells. These enzymes are known to play important and diverse roles in vesicular trafficking, DNA repair, cell cycle progression and cytokinesis in organisms from yeast to humans. In multicellular organisms they also regulate developmental pathways, control circadian rhythms, and have been implicated in Alzheimer's disease progression. Seven members of the CKI family have been identified (α, β, γ1, γ2, γ3, δ, and ε isoforms). It is likely that CKI isoforms also play an essential role in protozoan parasites since these enzymes have been described for Plasmodium, Leishmania and Trypanosoma parasites (Barik, S. et al., 1997, J. Biol. Chem. 272:26132-26138; Vieira, L. L. et al., 2002, Int. J. Parasitol. 32:1085-1093; Sacerdoti-Sierra, N. and Jaffe, C. L., 1997, J. Biol. Chem. 272:30760-30765; Spadafora, C. et al., 2002, Mol. Biochem. Parasitol. 124:23-36; Calabokis, M. et al., 2002, Parasitol. Int. 51:25-39). The present invention relates to the cloning, expression and characterization of three novel CKI enzymes, two isoforms from the species Toxoplasma gondii (TgCKIα and TgCKIβ) and one from the species Eimeria tenella (EtCKIα).
Affinity-ligand purification studies from Knockaert et al., 2000, supra, using an immobilized purvalanol column identified a single binding protein from T. gondii lysates. Microsequencing of an eleven amino acid peptide demonstrated that the Toxoplasma protein displayed sequence similarity to Arabidopsis thaliana CKI, and thus the protein was labeled Toxoplasma CKI.
Eimeria and Toxoplasma are related coccidian protozoa, a subgroup of the phylum Apicomplexa that includes intestinal parasites of veterinary and clinical significance. The poultry industry is most severely affected by Eimeria spp. infections resulting in coccidiosis. Worldwide costs of $800 million have been reported by the industry, encompassing the cost of prophylactic in-feed medications, alternative treatments if those medications fail, and losses due to mortality and poor feed conversions of infected birds (Allen, P. C. and Fetterer, R. H., 2002, Clin. Microbiol. Rev. 15:58-65). Anticoccidial compounds introduced nearly 30 years ago continue to be used prophylactically in poultry operations, the most successful being the polyether ionophores. However, reports of resistance to the current compounds are common due to the constant chemotherapeutic pressure exerted by this class of compounds. Toxoplasma gondii infects a broad range of warm-blooded animals. Although it is usually benign in humans, Toxoplasmosis can result in significant mortality and/or morbidity in congenital infections and immunocompromised patients. Current treatment for Toxoplasmosis is a combination therapy using pyrimethamine and sulfonamide; however, significant toxicity often accompanies this treatment regime. There is a current need for identification and development of new compounds for treatment of Toxoplasmosis and coccidiosis. To meet this end, the coccidian casein Kinase I enzymes disclosed herein represent novel targets for the development of broad-spectrum coccidiostat compounds effective against coccidiosis and Toxoplasmosis.